Elsevier

Brain Research

Volume 1303, 25 November 2009, Pages 151-160
Brain Research

Research Report
Thyrotropin-releasing hormone d,l polylactide nanoparticles (TRH-NPs) protect against glutamate toxicity in vitro and kindling development in vivo

https://doi.org/10.1016/j.brainres.2009.09.039Get rights and content

Abstract

Thyrotropin-releasing hormone (TRH) is reported to have anticonvulsant effects in animal seizure models and certain intractable epileptic patients. However, its duration of action is limited by rapid tissue metabolism and the blood brain barrier. Direct nose–brain delivery of neuropeptides in sustained-release biodegradable nanoparticles (NPs) is a promising mode of therapy for enhancing CNS bioavailability. Bioactivity/neuroprotection of d,l polylactide nanoparticles containing TRH was assessed against glutamate toxicity in cultured rat fetal hippocampal neurons. Subsequently, we utilized the kindling model of temporal lobe epilepsy to determine if intranasal administration of nanoparticles containing TRH (TRH-NPs) could inhibit kindling development. Animals received daily treatments of either blank (control) or TRH-NPs for 7 days before initiation of kindling. On day 8 and each day thereafter until either fully kindled or until day 20, the animals received daily treatments before receiving a kindling stimulus 3 h later. Afterdischarge duration (ADD) was assessed via electroencephalographs recorded from electrodes in the basolateral amygdalae and behavioral seizure stereotypy was simultaneously recorded digitally. Intranasal application of TRH-NPs resulted in a significant reduction in seizure ADD as kindling progressed, while the number of stimulations required to reach stage V seizures and to become permanently kindled was significantly greater in TRH-NP-treated subjects. Additionally, delay to clonus was significantly prolonged while clonus duration was reduced indicating a less severe seizure in TRH-NP-treated subjects. Our results provide proof of principle that intranasal delivery of sustained-release TRH-NPs may be neuroprotective and can be utilized to suppress seizures and perhaps epileptogenesis.

Introduction

Thyrotropin-releasing hormone (TRH) was the first hypothalamic releasing factor to be fully characterized for neuroendocrine control of the anterior pituitary (Boler et al., 1969, Guillemin, 1978, Schally, 1978). Subsequently, it was determined that as much as two thirds of the TRH in the brain is also widely distributed in extrahypothalamic structures throughout the CNS (Kreider et al., 1985, Kubek et al., 1977, Winokur and Utiger, 1974) where it is synthesized in neurons, stored and transported in vesicles, and released at synaptic terminals. Once in the synapse, it binds to specific TRH receptors on neurons and regulates the actions of several classical neurotransmitters, indicating a broad range of neuromodulatory functions (Nillni and Sevarino, 1999).

TRH has a notable presence in limbic system area (Kubek et al., 1977, Kubek, 1986, Manaker et al., 1985), which increases substantially following electroconvulsive and amygdala-kindled seizures (Knoblach and Kubek, 1994, Knoblach and Kubek, 1997a, Kubek and Sattin, 1984, Kubek et al., 1985, Pekary et al., 1997, Rosen et al., 1992). In addition, TRH has been shown to be anti-epileptic in a number of animal seizure models (Jaworska-Feil et al., 1999, Jaworska-Feil et al., 2001, Kubek et al., 1989, Kubek et al., 1993, Kubek et al., 1998, Kubek and Garg, 2002, Veronesi et al., 2007a, Wan et al., 1998). In clinical studies, TRH analogs were found to be efficacious against intractable seizures that occur in West Syndrome, Lennox–Gastaut Syndrome and other refractory epileptic syndromes (Inanaga et al., 1989, Matsuishi et al., 1983, Matsumoto et al., 1987, Matsumoto et al., 1989, Takeuchi et al., 1995, Takeuchi et al., 1999, Tanaka et al., 1998).

As a neuropeptide, TRH access to the CNS following oral or parenteral administration is limited by rapid degradation by specific and non-specific enzymes and difficulty in crossing the blood–brain barrier (BBB) (Garat et al., 1985, Heuer et al., 1998, Kastin and Pan, 2003). The intranasal route of delivery presents a possible alternative route of administration for TRH and other neuropeptides to access to the brain through the nasal cavity by way of transport through the olfactory neuroepithelium (Kubek et al., 2001, Kubek et al., 2007). This is not a new concept but the discovery of newer biodegradable nanoparticle formulations has generated renewed interest in this approach to CNS drug delivery. Presently we do not know the preferred route by which intranasally administered drugs and nanoparticles gain entry to the CNS from the olfactory epithelium. At least three possibilities exist: (1) olfactory nerve uptake; (2) paracellular uptake; or (3) microvasular uptake. Understandably, this approach is the subject of extensive ongoing research (see Lockman et al., 2002; and Kubek et al., 2007 for reviews). Kindling is a chronic, electrically-induced animal model of TLE, which mimics partial seizures and has been studied more extensively than any other temporal lobe epilepsy/seizure model (Bertram, 2007, Loscher, 1997). Recurrent, subconvulsive, electrical stimulation of certain brain structures, such as the amygdala or hippocampus, can reliably lead to kindled seizures (Goddard et al., 1969). Full kindling is permanent and includes behavioral, electrophysiological, neuroanatomical, and neurochemical characteristics associated with partial and generalized seizures. Limbic kindling begins with an initial focal seizure response at the stimulated site, which is expressed on the EEG as an epileptiform discharge. With repeated daily electrical stimulations, the afterdischarges become progressively more complex and prolonged until generalized tonic–clonic seizures (stage V) occur (Racine, 1972). After 3–5 consecutive stage V seizures, the animal is said to be permanently kindled (Goddard et al., 1969).

It was recently demonstrated that intranasal delivery of TRH/analogs significantly reduced several seizure parameters in fully kindled animals for up to an hour after administration (Chepurnov et al., 2002, Veronesi et al., 2007a). While the results demonstrated proof of principle for the intranasal route of TRH delivery to the CNS, it is apparent that sustained bioavailability is essential for a more durable therapeutic effect. Novel, sustained-release nanoparticle-sized biodegradable carriers may offer a plausible solution for enhancing TRH bioavailability by facilitating transport to the brain across membrane barriers (Kubek et al., 2001, Kubek et al., 2007). d,l Polylactide nanoparticles are submicron-sized (ideally  100 nm) polymeric colloidal particles, with a therapeutic agent of interest (i.e. TRH) entrapped within their polymeric matrix (Domb et al., 1991, Domb and Kubek, 2001, Langer, 1997). Reports have appeared showing intranasal uptake and delivery of nanoparticles to the brain but without demonstrating a subsequent physiological effect (Gao et al., 2006, Oberdorster et al., 2004, Zhang et al., 2006). Recently, we reviewed both in vitro and in vivo evidence in support of intranasal nanoparticle delivery to the brain (Kubek et al., 2009). Here we provide additional data showing TRH nanoparticle protection against glutamate toxicity in vitro, and that intranasal delivery of TRH nanoparticles are capable of suppressing a number of seizure characteristics in the rat kindling model of temporal lobe epilepsy. We first reported this data in abstract form (Veronesi et al., 2008).

Section snippets

Results

Poly (l-lactic acid–d-lactic acid) nanoparticles with or without TRH were prepared by the solvent evaporation method using a double emulsion process. TRH-loaded nanoparticle (TRH-NPs) samples measured by the particle analyzer as well as electron microscopy (TEM) revealed a mean diameter of 108 +/− 12 nm for the TRH-NPs and 102 +/− 12 nm for the blank-NPs demonstrating a uniformity of nanoparticle size and shape. Cultured hippocampal neurons treated with 500 μM Glu alone for 18 h resulted in a

Discussion

The use of TRH-NPs is hypothesized to increase target tissue bioavailability following intranasal delivery. However, little is known concerning the characteristics of TRH-NPs during release or its bioactivity following release. We have shown previously that TRH/analogs are effective in protecting cultured, primary fetal hippocampal neurons against 500 μM Glu toxicity in a dose-dependent manner and that high concentrations of TRH are not toxic to cultured neurons (Veronesi et al., 2007b).

Preparation of d,l PLA nanoparticles loaded with TRH

l- and d-Lactide were obtained from Purac BV (Gorinchem, Holland). Dichloromethane (DCM) and other solvents were HPLC grade and purchased from BioLab Ltd. (Jerusalem, Israel). All other materials and agents were ordered from Sigma-Aldrich (Rehovot, Israel). Poly (l-lactic acid-d-lactic acid) (d,l-PLA) was prepared by ring-opening polymerization of l-lactide and d-lactide using stannous 1-ethylhexanoate (Sn(Oct)2) as a catalyst and benzyl alcohol as a co-catalyst. The synthesis and polymer

Conflict of interest statement

None of the authors has any conflict of interest to disclose.

Acknowledgments

This work was sponsored by a grant from Citizens United for Research in Epilepsy (CURE). The authors would like to thank Mr. Daniel Kubek, Miss Amanda Weinert, Dr. William Truitt and Ms. Amy Kostrewza for their expert technical assistance. Dr. Vincent Gattone and Ms. Caroline Miller of the Indiana University School of Medicine Electron Microscopy Center were instrumental in helping us characterize the TRH-NPs. We thank Ms. Cynthia Cally, Division of Biostatistics, Department of Medicine for

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